In order to analyze the functions of proteins, two-dimensional electrophoresis has long been used. But recently, proteomics, a post-genome functional analyzing method, took the place of it, which was led by the development of mass spectrometer like MALDI-TOF and the establishment of easy methods for determining N-terminal amino acid sequences. The proteomics, though, is limited in use for the research of cancer caused by the result of highly complicated signal transduction, because proteomics is selectively available for the analysis of functions at one point of time although the human body is in dynamic motion. In order to detect a cancer, it is more important to investigate the increased expression or post-translational modification that are the result of signal transduction than to observe the appearance of new spots by staining. The amounts of proteins that can be detected by two-dimensional electrophoresis are too small to analyze by simple staining. Observing glycosylation of proteins can help overcome the above problem and correct the analytical errors based on post-translational modification. When a patient group with cancer was compared with a control group, it was difficult to find out the difference of spots between the two groups by general electrophoresis, in the meantime, it was possible to draw a clear line between the two groups by analyzing the changes of glycosylation with lectin. It is now called glycomics, that is, an upgraded analytical method overcoming difficulties in proteomics analysis characterized by tracing the changes of glycosylation of proteins while post-translational modification is occurring.
From observing cellular biological changes during tumorigenesis and metastasis processes, it is concluded that various kinds of glycoproteins or glycolipids on the surface of cell membrane are induced to go through “aberrant glycosylation” by the specific signal from oncogene, resulting in the changes of sugar chain that consecutively causes the changes of intercellular adhesion and recognition, resulting in tumorigenesis and metastasis in the event (Hakomori and Kannagi, 1983, J. Natl. Cancer Inst., 71:231-251; Feizi, 1985, Nature, 314:53-571). When external stimuli come in, signals are transmitted via oncogene ras, transcription factor ets-1 to stimulate the expression of N-acetylglucosaminyltransferase V (GnT-V). GnT-V is an enzyme catalyzing a reaction attaching N-acetylglucosamine onto the β1,6 site of the basic sugar chain of a glycoprotein and is known to be directly associated with cancer invasion and metastasis (Dennis, et al., 1987, Science, 236:582-585). As for glycoproteins, basic sugar chain is formed in endoplasmic reticulum (ER) after a protein is synthesized, which moves to the Golgi apparatus. Then, sugars are added thereto by various glycotransferases resulted from various vital phenomena of cells. Primary sugar chains are formed by catalyzing of six N-acetylglucosaminyltransferases (I-VI). GnT-V, which forms β1,6-N-acetylglucosamine sugar chain, has been believed to be deeply associated with tumorigenesis and metastasis. GnT-V is located in Golgi apparatus. This enzyme makes target proteins be secreted to or out of the cell surface by causing the changes of sugar chains. At this time, glycoproteins recognize surface proteins of target cells and then adhere thereto, causing a cancer.
Dennis et al. (Science 236(4801): 582-585, 1987) first reported that the β1,6 branches were remarkably represented as cancer tissues were growing or during metastasis. A cell surface protein gp130 is one of the major target proteins of GnT-V and shows highly metastasis activity when β1,6 N-acetylglucosamine is added. GnT-V knockout mice were established using embryonic stem cells in which GnT-V was deficient and to which polyomavirus middle T antigen (referred “PyMT” hereinafter) viral oncogene was introduced in order to induce a cancer. As a result, the growth of cancer and metastasis induced by PyMT were remarkably inhibited in GnT-V knockout mice comparing with another normal mice group in which only PyMT was over-expressed (Granovsky, et al., 2000, Nature Med., 6:306-312), and the growth of β1,6 branch caused high metastasis especially in mice with breast cancer. Recent studies support that the GnT-V activity of 33 types of hepatocellular carcinoma (HCC) tissues is fifty times as high as it's activity to normal tissues and four times as high as that of cancer surrounding tissues (Yao, et al., 1998, J Cancer Res. Clin. Oncol., 124:27-307). And high metastatic activity was also confirmed when large intestine cancer cell line WiDr in which GnT-V was over-expressed was injected into immunodeficient mice in order to induce large intestine cancer or when angiogenesis was investigated by CAM analysis using fertilized eggs (Miyoshi, et al., 2001, unpublished results). Thus, GnT-V is believed to be associated with metastasis and have high metastatic activity regardless of types of tissues. GnT-V enzyme was purified from human lung cancer cells and a mouse kidney, and cDNA cloning and analysis of genomic structure and promoter of the enzyme have been made (Gu, et al., 1993, J. Biochem, 113:614-619; Soreibah, et al., 1993, J. Biol. Chem., 268:15381-15385; Kang, et al., 1996, J. Biol. Chem., 271:26706-26712). The present inventors also reported in a recent study that transcription factor ets-1 was deeply associated with the expression of GnT-V (Ko, et al., 1999, J. Biol. Chem., 274(33):22941-22948). As for large intestine cancer, it now takes the 4th highest incidence in both men and women owing to the changes of dietary life into western and the development of the cancer increases continuously. However, there is no way to diagnose colon cancer accurately except large intestine endoscopy, so far.
Thus, the present inventors detected β1,6-N-acetylglucosamine in which sugars were attached by GnT-V in cancer-induced cells and found out a novel glycoprotein showing the changes of sugar chains by analyzing amino acid sequences with a mass spectrometer. And the present inventors completed this invention by developing a method for diagnosing cancers by measuring the changes of sugar chains of the above protein in test samples and a diagnostic kit for cancers using the same.